Advertisement

Nasal Administration of Cationic Nanoemulsions as CD73-siRNA Delivery System for Glioblastoma Treatment: a New Therapeutical Approach

  • J. H. AzambujaEmail author
  • R. S. Schuh
  • L. R. Michels
  • N. E. Gelsleichter
  • L. R. Beckenkamp
  • I. C. Iser
  • G. S. Lenz
  • F. H. de Oliveira
  • G. Venturin
  • S. Greggio
  • J. C. daCosta
  • M. R. Wink
  • J. Sevigny
  • M. A. Stefani
  • A. M. O. Battastini
  • H. F. Teixeira
  • E. Braganhol
Article
  • 31 Downloads

Abstract

Glioblastoma is the most devastating primary brain tumor. Effective therapies are not available, mainly due to high tumor heterogeneity, chemoresistance, and the difficulties imposed by blood-brain barrier. CD73, an enzyme responsible for adenosine (ADO) production, is overexpressed in cancer cells and emerges as a target for glioblastoma treatment. Indeed, ADO causes a variety of tumor-promoting actions, particularly by inducing tumor immune escape, whereas CD73 inhibition impairs tumor progression. Here, a cationic nanoemulsion to deliver CD73siRNA (NE-siRNA CD73R) via nasal route aiming glioblastoma treatment was developed. NE-siRNA CD73R was uptaken by glioma cells in culture, resulting in a parallel 60–80% decrease in AMPase activity and 30–50% in cell viability. Upon nasal delivery, NE-siRNA CD73R was detected in rat brain and serum. Notably, treatment with CD73siRNA complexes of glioma-bearing Wistar rats reduced tumor growth by 60%. Additionally, NE-siRNA CD73R treatment decreased 95% ADO levels in liquor and tumor CD73 expression, confirming in vivo CD73 silencing. Finally, no toxicity was observed in either primary astrocytes or rats with this cationic nanoemulsion. These results suggest that nasal administration of cationic NE as CD73 siRNA delivery system represents a novel potential treatment for glioblastoma.

Graphical Abstract

Glioblastoma is the most common and devastating form of primary brain tumor. CD73, a protein involved in cell-cell adhesion and migration processes and also responsible for extracellular adenosine (ADO) production, is overexpressed by glioma cells and emerges as an important target for glioma treatment. Indeed, ADO participates in tumor immune escape, cell proliferation, and angiogenesis, and CD73 inhibition impairs those processes. Here, a cationic nanoemulsion to deliver CD73 siRNA (NE-siRNA CD73R) via nasal route aiming glioblastoma treatment was developed. NE-siRNA CD73R knockdown in vitro and in vivo CD73. Upon nasal delivery of NE-siRNA CD73R, the treatment markedly reduced tumor volume by 60% in a rat preclinical glioblastoma model. The treatment was well tolerated, and did not induce kidney, liver, lung, olfactory, bone marrow, or behavior alterations. These results indicate that the nasal administration of NE as a CD73 siRNA delivery system offered an efficient means of gene knockdown and may represent a potential alternative for glioblastoma treatment.

Keywords

Adenosine Brain delivery Cationic nanoemulsion CD73 Glioma 

Abbreviations

ADO

adenosine

ATCC

American Type Culture Collections

BBB

Blood-brain barrier

CD73

Ecto-5′-nucleotidase

CNS

Central nervous system

CSF

Cerebrospinal fluid

DAPI

4′, 6-diamidino-2-phenylindole, dihydrochloride

DMEM

Dulbecco’s Modified Eagle Medium

DOTAP

1,2-dioleoyl-sn-glycero-3-trimethylammonium propane

FBS

Fetal bovine serum

HPCL

High-performance liquid chromatography

INO

Inosine

NE

Nanoemulsion

NE-siRNA CD73R

nanoemulsion loaded rat CD73 siRNA

NE-siRNA CD73H

nanoemulsion loaded human CD73 siRNA

NE-siRNA scramble

nanoemulsion loaded GFP siRNA

NR-NE

Nile red label nanoemulsion

MCT

Medium chain triglycerides

PCS

Photon correlation spectroscopy

siRNA

Small interfering ribonucleic acids

RT

Room temperature

TCA

Trichloroacetic acid

TMZ

Temozolomide

Notes

Funding Information

This study was supported by the Brazilian agencies: Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq — Processo 422298/2016-6; 310846/2014-5), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS — Processo 16/2551-0000265-7; PRONEX —Processo 16/2551-0000473-0); J.H. Azambuja, R.S. Schuh, N.E. Gelsleichter, L.R. Beckenkamp, G.S. Lenz, da Costa, J.C. were recipients of UFCSPA, CAPES, or CNPq fellowships. J.S. received support from the Canadian Institutes of Health Research (CIHR) and was the recipient of a “Chercheur National” Scholarship from the Fonds de Recherche du Québec – Santé (FRQS).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflicts of interest.

Supplementary material

12035_2019_1730_MOESM1_ESM.jpg (86 kb)
ESM 1 Methodology used in the olfactory test (JPG 86 kb)
12035_2019_1730_Fig8_ESM.png (161 kb)
ESM 2

NE-siRNA CD73R complexes did not change AMPase activity in non-transformed cells. Hacat cells (human keratinocyte cell line) were exposed to NE-siRNA scramble or NE-siRNA CD73H at charge ratios +2/−1 and + 4/−1 for 48 h, as indicated. Analysis of AMPase activity in Hacat healthy cells was performed by malachite green method. Data represent mean ± SD of at least three independent experiments performed in triplicate. Data were analyzed by ANOVA followed by Tukey post hoc. NE-siRNA scramble = nanoemulsion complexed with GFP siRNA; NE-siRNA CD73R = nanoemulsion complexed with rat CD73 siRNA. (PNG 161 kb)

12035_2019_1730_MOESM2_ESM.tif (1.5 mb)
High Resolution Image (TIF 1486 kb)

References

  1. 1.
    Ostrom QT, Bauchet L, Davis FG, Deltour I, Fisher JL, Langer CE, Pekmezci M, Schwartzbaum JA, Turner MC, Walsh KM, Wrensch MR, Barnholtz-Sloan JS (2014) The epidemiology of glioma in adults: a “state of the science”; review. Neuro-Oncology 16:896–913.  https://doi.org/10.1093/neuonc/nou087
  2. 2.
    Lara-Velazquez M, Al-Kharboosh R, Jeanneret S et al (2017) Advances in brain tumor surgery for glioblastoma in adults. Brain Sci 7:166.  https://doi.org/10.3390/brainsci7120166 CrossRefGoogle Scholar
  3. 3.
    Wijaya J, Fukuda Y, Schuetz JD (2017) Obstacles to brain tumor therapy: key ABC transporters. Int J Mol Sci 18.  https://doi.org/10.3390/ijms18122544
  4. 4.
    Schiera G, Maria Di Liegro C, Di Liegro I (2017) Molecular sciences molecular determinants of malignant brain cancers: from intracellular alterations to invasion mediated by extracellular vesicles.  https://doi.org/10.3390/ijms18122774
  5. 5.
    Stupp R, van den Bent MJ, Hegi ME (2005) Optimal role of temozolomide in the treatment of malignant gliomas. Curr Neurol Neurosci Rep 5:198–206CrossRefGoogle Scholar
  6. 6.
    Wang Z, Yang G, Zhang Y-Y, Yao Y, Dong LH (2017) A comparison between oral chemotherapy combined with radiotherapy and radiotherapy for newly diagnosed glioblastoma: a systematic review and meta-analysis. Medicine (Baltimore) 96:e8444.  https://doi.org/10.1097/MD.0000000000008444 CrossRefGoogle Scholar
  7. 7.
    Wang W, Liu F, Xiang B, Xiang C, Mou X (2015) Stem cells as cellular vehicles for gene therapy against glioblastoma. Int J Clin Exp Med 8:17102–17109Google Scholar
  8. 8.
    Glaser T, Han I, Wu L, Zeng X (2017) Targeted nanotechnology in glioblastoma multiforme. Front Pharmacol 8(166).  https://doi.org/10.3389/fphar.2017.00166
  9. 9.
    Zheng M, Tao W, Zou Y, Farokhzad OC, Shi B (2018) Nanotechnology-based strategies for siRNA brain delivery for disease therapy. Trends Biotechnol 36:562–575.  https://doi.org/10.1016/j.tibtech.2018.01.006 CrossRefGoogle Scholar
  10. 10.
    Azambuja JH, Gelsleichter NE, Beckenkamp LR, Iser IC, Fernandes MC, Figueiró F, Battastini AMO, Scholl JN et al (2018) CD73 downregulation decreases in vitro and in vivo glioblastoma growth. Mol Neurobiol 56:3260–3279.  https://doi.org/10.1007/s12035-018-1240-4 CrossRefGoogle Scholar
  11. 11.
    Quezada C, Garrido W, Oyarzún C, Fernández K, Segura R, Melo R, Casanello P, Sobrevia L et al (2013) 5′-ectonucleotidase mediates multiple-drug resistance in glioblastoma multiforme cells. J Cell Physiol 228:602–608.  https://doi.org/10.1002/jcp.24168 CrossRefGoogle Scholar
  12. 12.
    Kazemi MH, Raoofi Mohseni S, Hojjat-Farsangi M, Anvari E, Ghalamfarsa G, Mohammadi H, Jadidi-Niaragh F (2018) Adenosine and adenosine receptors in the immunopathogenesis and treatment of cancer. J Cell Physiol 233:2032–2057.  https://doi.org/10.1002/jcp.25873 CrossRefGoogle Scholar
  13. 13.
    Antonioli L, Novitskiy SV, Sachsenmeier KF, Fornai M, Blandizzi C, Haskó G (2017) Switching off CD73: a way to boost the activity of conventional and targeted antineoplastic therapies. Drug Discov Today 22:1686–1696.  https://doi.org/10.1016/j.drudis.2017.06.005 CrossRefGoogle Scholar
  14. 14.
    Vijayan D, Young A, Teng MWL, Smyth MJ (2017) Targeting immunosuppressive adenosine in cancer. Nat Rev Cancer 17:765–765.  https://doi.org/10.1038/nrc.2017.110 CrossRefGoogle Scholar
  15. 15.
    Allard B, Beavis PA, Darcy PK, Stagg J (2016) Immunosuppressive activities of adenosine in cancer. Curr Opin Pharmacol 29:7–16.  https://doi.org/10.1016/j.coph.2016.04.001 CrossRefGoogle Scholar
  16. 16.
    Allard D, Chrobak P, Allard B, Messaoudi N, Stagg J (2018) Targeting the CD73-adenosine axis in immuno-oncology. Immunol Lett 205:31–39.  https://doi.org/10.1016/j.imlet.2018.05.001 CrossRefGoogle Scholar
  17. 17.
    Leone RD, Emens LA (2018) Targeting adenosine for cancer immunotherapy. J Immunother Cancer 6:57.  https://doi.org/10.1186/s40425-018-0360-8 CrossRefGoogle Scholar
  18. 18.
    Bavaresco L, Bernardi A, Braganhol E, Cappellari AR, Rockenbach L, Farias PF, Wink MR, Delgado-Cañedo A et al (2008) The role of ecto-5′-nucleotidase/CD73 in glioma cell line proliferation. Mol Cell Biochem 319:61–68.  https://doi.org/10.1007/s11010-008-9877-3 CrossRefGoogle Scholar
  19. 19.
    Cappellari AR, Vasques GJ, Bavaresco L, Braganhol E, Battastini AMO (2012) Involvement of ecto-5′-nucleotidase/CD73 in U138MG glioma cell adhesion. Mol Cell Biochem 359:315–322.  https://doi.org/10.1007/s11010-011-1025-9 CrossRefGoogle Scholar
  20. 20.
    Jadidi-Niaragh F, Atyabi F, Rastegari A, Kheshtchin N, Arab S, Hassannia H, Ajami M, Mirsanei Z et al (2017) CD73 specific siRNA loaded chitosan lactate nanoparticles potentiate the antitumor effect of a dendritic cell vaccine in 4T1 breast cancer bearing mice. J Control Release 246:46–59.  https://doi.org/10.1016/j.jconrel.2016.12.012 CrossRefGoogle Scholar
  21. 21.
    Terp MG, Olesen KA, Arnspang EC, Lund RR, Lagerholm BC, Ditzel HJ, Leth-Larsen R (2013) Anti-human CD73 monoclonal antibody inhibits metastasis formation in human breast Cancer by inducing clustering and internalization of CD73 expressed on the surface of cancer cells. J Immunol 191:4165–4173.  https://doi.org/10.4049/jimmunol.1301274 CrossRefGoogle Scholar
  22. 22.
    Zhi X, Chen S, Zhou P, Shao Z, Wang L, Ou Z, Yin L (2007) RNA interference of ecto-5′-nucleotidase (CD73) inhibits human breast cancer cell growth and invasion. Clin Exp Metastasis 24:439–448.  https://doi.org/10.1007/s10585-007-9081-y CrossRefGoogle Scholar
  23. 23.
    Zhi X, Wang Y, Zhou X, Yu J, Jian R, Tang S, Yin L, Zhou P (2010) RNAi-mediated CD73 suppression induces apoptosis and cell-cycle arrest in human breast cancer cells. Cancer Sci 101:2561–2569.  https://doi.org/10.1111/j.1349-7006.2010.01733.x CrossRefGoogle Scholar
  24. 24.
    Tekade RK, Tekade M, Kesharwani P, D’Emanuele A (2016) RNAi-combined nano-chemotherapeutics to tackle resistant tumors. Drug Discov Today 21:1761–1774.  https://doi.org/10.1016/j.drudis.2016.06.029 CrossRefGoogle Scholar
  25. 25.
    Malhotra M, Toulouse A, Godinho BMDC, Mc Carthy DJ, Cryan JF, O'Driscoll CM (2015) RNAi therapeutics for brain cancer: current advancements in RNAi delivery strategies. Mol BioSyst 11:2635–2657.  https://doi.org/10.1039/C5MB00278H CrossRefGoogle Scholar
  26. 26.
    Messaoudi K, Clavreul A, Lagarce F (2015) Toward an effective strategy in glioblastoma treatment. Part II: RNA interference as a promising way to sensitize glioblastomas to temozolomide. Drug Discov Today 20:772–779.  https://doi.org/10.1016/j.drudis.2015.02.014 CrossRefGoogle Scholar
  27. 27.
    Buduru S, Zimta A-A, Ciocan C, Braicu C, Dudea D, Irimie AI, Berindan-Neagoe I (2018) RNA interference: new mechanistic and biochemical insights with application in oral cancer therapy. Int J Nanomedicine 13:3397–3409.  https://doi.org/10.2147/IJN.S167383 CrossRefGoogle Scholar
  28. 28.
    Jain S, Pathak K, Vaidya A (2018) Molecular therapy using siRNA: recent trends and advances of multi target inhibition of cancer growth. Int J Biol Macromol 116:880–892.  https://doi.org/10.1016/j.ijbiomac.2018.05.077 CrossRefGoogle Scholar
  29. 29.
    Karim ME, Tha KK, Othman I, Borhan Uddin M, Chowdhury E (2018) Therapeutic potency of nanoformulations of siRNAs and shRNAs in animal models of cancers. Pharmaceutics 10.  https://doi.org/10.3390/pharmaceutics10020065
  30. 30.
    Kumar A, Pandey AN, Jain SK (2016) Nasal-nanotechnology: revolution for efficient therapeutics delivery. Drug Deliv 23:681–693.  https://doi.org/10.3109/10717544.2014.920431 Google Scholar
  31. 31.
    Crowe TP, Greenlee MHW, Kanthasamy AG, Hsu WH (2018) Mechanism of intranasal drug delivery directly to the brain. Life Sci 195:44–52.  https://doi.org/10.1016/j.lfs.2017.12.025 CrossRefGoogle Scholar
  32. 32.
    Pires PC, Santos AO (2018) Nanosystems in nose-to-brain drug delivery: a review of non-clinical brain targeting studies. J Control Release 270:89–100.  https://doi.org/10.1016/j.jconrel.2017.11.047 CrossRefGoogle Scholar
  33. 33.
    Bourganis V, Kammona O, Alexopoulos A, Kiparissides C (2018) Recent advances in carrier mediated nose-to-brain delivery of pharmaceutics. Eur J Pharm Biopharm 128:337–362.  https://doi.org/10.1016/j.ejpb.2018.05.009 CrossRefGoogle Scholar
  34. 34.
    Bahadur S, Pathak K (2012) Physicochemical and physiological considerations for efficient nose-to-brain targeting. Expert Opin Drug Deliv 9:19–31.  https://doi.org/10.1517/17425247.2012.636801 CrossRefGoogle Scholar
  35. 35.
    Fonseca FN, Betti AH, Carvalho FC, Gremião MPD, Dimer FA, Guterres SS, Tebaldi ML, Rates SMK et al (2015) Mucoadhesive amphiphilic methacrylic copolymer-functionalized poly(ε-caprolactone) nanocapsules for nose-to-brain delivery of olanzapine. J Biomed Nanotechnol 11:1472–1481CrossRefGoogle Scholar
  36. 36.
    Khan AR, Liu M, Khan MW, Zhai G (2017) Progress in brain targeting drug delivery system by nasal route. J Control Release 268:364–389.  https://doi.org/10.1016/j.jconrel.2017.09.001 CrossRefGoogle Scholar
  37. 37.
    Teixeira HF, Bruxel F, Fraga M, Schuh RS, Zorzi GK, Matte U, Fattal E (2017) Cationic nanoemulsions as nucleic acids delivery systems. Int J Pharm 534:356–367.  https://doi.org/10.1016/j.ijpharm.2017.10.030 CrossRefGoogle Scholar
  38. 38.
    Singh Y, Meher JG, Raval K, Khan FA, Chaurasia M, Jain NK, Chourasia MK (2017) Nanoemulsion: concepts, development and applications in drug delivery. J Control Release 252:28–49.  https://doi.org/10.1016/j.jconrel.2017.03.008 CrossRefGoogle Scholar
  39. 39.
    Ganta S, Talekar M, Singh A, Coleman TP, Amiji MM (2014) Nanoemulsions in translational research-opportunities and challenges in targeted cancer therapy. AAPS PharmSciTech 15:694–708.  https://doi.org/10.1208/s12249-014-0088-9 CrossRefGoogle Scholar
  40. 40.
    Schuh RS, de Carvalho TG, Giugliani R, Matte U, Baldo G, Teixeira HF (2018) Gene editing of MPS I human fibroblasts by co-delivery of a CRISPR/Cas9 plasmid and a donor oligonucleotide using nanoemulsions as nonviral carriers. Eur J Pharm Biopharm 122:158–166.  https://doi.org/10.1016/j.ejpb.2017.10.017 CrossRefGoogle Scholar
  41. 41.
    Azambuja JH, da Silveira EF, de Carvalho TR, Oliveira PS, Pacheco S, do Couto CT, Beira FT, Stefanello FM et al (2017) Glioma sensitive or chemoresistant to temozolomide differentially modulate macrophage protumor activities. Biochim Biophys Acta, Gen Subj 1861:2652–2662.  https://doi.org/10.1016/j.bbagen.2017.07.007 CrossRefGoogle Scholar
  42. 42.
    Dos Santos LM, da Silva TM, Azambuja JH et al (2016) Methionine and methionine sulfoxide treatment induces M1/classical macrophage polarization and modulates oxidative stress and purinergic signaling parameters. Mol Cell Biochem 424:69–78.  https://doi.org/10.1007/s11010-016-2843-6 CrossRefGoogle Scholar
  43. 43.
    Chan KM, Delfert D, Junger KD (1986) A direct colorimetric assay for Ca2+-stimulated ATPase activity. Anal Biochem 157:375–380CrossRefGoogle Scholar
  44. 44.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  45. 45.
    Pacheco SM, Soares MSP, Gutierres JM, Gerzson MFB, Carvalho FB, Azambuja JH, Schetinger MRC, Stefanello FM et al (2018) Anthocyanins as a potential pharmacological agent to manage memory deficit, oxidative stress and alterations in ion pump activity induced by experimental sporadic dementia of Alzheimer’s type. J Nutr Biochem 56:193–204.  https://doi.org/10.1016/j.jnutbio.2018.02.014 CrossRefGoogle Scholar
  46. 46.
    Da Silveira EF, Chassot JM, Teixeira FC et al (2013) Ketoprofen-loaded polymeric nanocapsules selectively inhibit cancer cell growth in vitro and in preclinical model of glioblastoma multiforme. Investig New Drugs 31:1424–1435.  https://doi.org/10.1007/s10637-013-0016-y CrossRefGoogle Scholar
  47. 47.
    Fraga M, de Carvalho TG, Diel D d S et al (2015) Cationic nanoemulsions as a gene delivery system: proof of concept in the mucopolysaccharidosis I murine model. J Nanosci Nanotechnol 15:810–816CrossRefGoogle Scholar
  48. 48.
    Bruxel F, Bochot A, Diel D, Wild L, Carvalho E, Cojean S, Loiseau P, Fattal E et al (2014) Adsorption of antisense oligonucleotides targeting malarial topoisomerase II on cationic nanoemulsions optimized by a full factorial design. Curr Top Med Chem 14:1161–1171CrossRefGoogle Scholar
  49. 49.
    Yadav S, Gandham SK, Panicucci R, Amiji MM (2016) Intranasal brain delivery of cationic nanoemulsion-encapsulated TNFα siRNA in prevention of experimental neuroinflammation. Nanomedicine 12:987–1002.  https://doi.org/10.1016/j.nano.2015.12.374 CrossRefGoogle Scholar
  50. 50.
    Martini E, Fattal E, de Oliveira MC, Teixeira H (2008) Effect of cationic lipid composition on properties of oligonucleotide/emulsion complexes: physico-chemical and release studies. Int J Pharm 352:280–286.  https://doi.org/10.1016/j.ijpharm.2007.10.032 CrossRefGoogle Scholar
  51. 51.
    Teixeira H, Fraga F, Bruxel VL et al (2011) Influence of phospholipid composition on cationic emulsions/DNA complexes: physicochemical properties, cytotoxicity, and transfection on Hep G2 cells. Int J Nanomedicine 6:2213.  https://doi.org/10.2147/IJN.S22335 CrossRefGoogle Scholar
  52. 52.
    Koszałka P, Gołuńska M, Stanisławowski M, Urban A, Stasiłojć G, Majewski M, Wierzbicki P, Składanowski AC et al (2015) CD73 on B16F10 melanoma cells in CD73-deficient mice promotes tumor growth, angiogenesis, neovascularization, macrophage infiltration and metastasis. Int J Biochem Cell Biol 69:1–10.  https://doi.org/10.1016/j.biocel.2015.10.00354 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • J. H. Azambuja
    • 1
    Email author
  • R. S. Schuh
    • 2
  • L. R. Michels
    • 2
  • N. E. Gelsleichter
    • 1
  • L. R. Beckenkamp
    • 1
  • I. C. Iser
    • 1
  • G. S. Lenz
    • 1
  • F. H. de Oliveira
    • 3
  • G. Venturin
    • 4
  • S. Greggio
    • 4
  • J. C. daCosta
    • 4
  • M. R. Wink
    • 1
  • J. Sevigny
    • 5
    • 6
  • M. A. Stefani
    • 7
  • A. M. O. Battastini
    • 8
  • H. F. Teixeira
    • 2
  • E. Braganhol
    • 1
  1. 1.Programa de Pós-Graduação em BiociênciasUniversidade Federal de Ciências da Saúde de Porto Alegre (UFCSPA)Porto AlegreBrazil
  2. 2.Programa de Pós-Graduação em Ciências FarmacêuticasUniversidade Federal do Rio Grande do Sul (UFRGS)Porto AlegreBrazil
  3. 3.Departamento de PatologiaPorto AlegreBrazil
  4. 4.Brain Institute of Rio Grande do Sul (BraIns)Pontifical Catholic University of Rio Grande do Sul (PUCRS)Porto AlegreBrazil
  5. 5.Département de Microbiologie-Infectiologie et d’Immunologie, Faculté de MédecineUniversité LavalQCCanada
  6. 6.Centre de recherche du CHU de QuébecUniversité LavalQuébec CityCanada
  7. 7.Departamento de MorfologiaUFRGSPorto AlegreBrazil
  8. 8.Departamento de BioquímicaUFRGSPorto AlegreBrazil

Personalised recommendations